rAAV Gene Therapy in a Canavan's Disease Mouse Model Reveals Immune Impairments and an Extended Pathology Beyond the Central Nervous System.

Aspartoacylase (AspA) gene mutations cause the pediatric lethal neurodegenerative Canavan disease (CD). There is emerging promise of successful gene therapy for CD using recombinant adeno-associated viruses (rAAVs). Here, we report an intracerebroventricularly delivered AspA gene therapy regime using three serotypes of rAAVs at a 20-fold reduced dose than previously described in AspA(-/-) mice, a bona-fide mouse model of CD. Interestingly, central nervous system (CNS)-restricted therapy prolonged survival over systemic therapy in CD mice but failed to sustain motor functions seen in systemically treated mice. Importantly, we reveal through histological and functional examination of untreated CD mice that AspA deficiency in peripheral tissues causes morphological and functional abnormalities in this heretofore CNS-defined disorder. We demonstrate for the first time that AspA deficiency, possibly through excessive N-acetyl aspartic acid accumulation, elicits both a peripheral and CNS immune response in CD mice. Our data establish a role for peripheral tissues in CD pathology and serve to aid the development of more efficacious and sustained gene therapy for this disease.

Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10.

Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.

A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS Gene therapy in Canavan mice.

Canavan's disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤ 1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.

Gene transfer in the liver using recombinant adeno-associated virus.

Liver-directed gene transfer and gene therapy are rapidly gaining attention primarily because the liver is centrally involved in a variety of metabolic functions that are affected in various inherited disorders. Recombinant adeno-associated virus (rAAV) is a popular gene delivery vehicle for gene therapy, and intravenous delivery of some rAAV serotypes results in very efficient transduction in the liver. rAAV-mediated gene transfer to the liver can be used to create somatic transgenic animals or disease models for studying the function of various genes and miRNAs. The liver is the target tissue for gene therapy of many inborn metabolic diseases and may also be exploited as a "biofactory" for production of coagulation factors, insulin, growth hormones, and other non-hepatic proteins. Hence, efficient delivery of transgenes and small RNAs to the liver by rAAV vectors has been of long-standing interest to research scientists and clinicians alike. This unit describes methods for delivery of rAAV vectors by several injection routes, followed by a range of analytical methods for assessing the expression, activity, and effects of the transgene and its product.

Several rAAV vectors efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system.

Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood-brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.